MXPA02002639A - Antibody dye conjugates for binding to target structures of angiogenesis in order to intraoperatively depict tumor peripheries. - Google Patents

Abstract

The invention relates to antibody dye conjugates which are suited for binding to structures of newly formed vessels and to the their use for interoperatively depicting pathological angiogenesis.

Description

CONJUGATES OF COLORANT AND ANTIBODY FOR ANGIOGENIC DIANE STRUCTURES, FOR INTRAOPERATIVE VISUALIZATION VAT OF THE TUMOR EDGE DESCRIPTION OF THE INVENTION This invention relates to colorant and antibody conjugates which are suitable for bonding to newly formed vessel structures, and to their use for intraoperative visualization of pathological angiogenesis. With some exceptions, the formation of new vessels does not take place in the adult organism (for example, the cycle of women of gestational age). However, it is possible to observe the new formation of vessels in many diseases. The process of the new formation of vessels that takes place there is called angiogenesis, and develops in response to certain signals. Angiogenesis is a process that preferably develops in the edge area of a disease focus. Factors that diffuse to the edge area of the disease focus are released from the center of the disease focus. These factors are also called angiogenesis stimulators. If these stimulators of angiogenesis reach healthy tissue in the edge area of the disease focus, vessels previously not integrated into the focus of the disease are stimulated to form new vascular buds. These vessels that extend from these vascular buds form a new capillary vascular network in the border area of the disease focus. Through this process you can always ensure an adequate supply of nutrients for the focus of the disease. It has been found to be particularly important that the growth of tumors and their metastases depends on the ability to induce angiogenesis. Surgical therapy is now a standard measure for treating localized foci of disease. It has acquired great importance in the case of the treatment of tumors. However, it turns out that despite the improvement of surgical techniques, the number of local recurrences is considerable because the anatomical conditions of the human organism only rarely allow a large-scale removal of the focus of the disease. In many organs (for example, in the brain) large-scale removal must be eliminated to obtain healthy tissue. The risk of injuring healthy organs increases with the degree of radicality of the surgical intervention. Histological studies of the area of the edge of the tumor after completion of tumor removal by surgery show, however, that a considerable number of tumors can not be completely removed and that radicals of the tumor remain in the body. Additional tumor growth and tumor metastasis can arise from these tumor radicals. A process that would exactly indicate the limits of a disease process with respect to healthy tissue during surgical treatment would allow the total removal of the focus of the disease and leave healthy tissue largely unaffected. Colorants for the visualization of disease foci are already known (Poon, W. S. et al., J. Neurosurgery (1992) 76: 679-686, Haglund, M. M. et al., Neurosurgery (1996) 38: 308-317). Preferably they are removed directly from the tumor cells or accumulate in non-specific form in the extracellular space of the tumors. Since the mechanism can be detected in concentration even in healthy tissue, the specificity and sensitivity of the substances used is low. To date there are no known compounds that can be used for the intraoperative delineation of disease foci by selective visualization of the edge area of a disease focus. Angiogenesis preferably takes place in the edge area of the disease foci. By visualizing angiogenesis you can visualize the boundary with healthy tissue. Antibodies to detect angiogenesis in the focus of the disease are already known and are used to visualize newly formed vessels in the histological section of the tissue, to detect several proteins in the focus of the disease or as carrier molecules for therapeutic substances. However, antibodies are not known in combination with dyes, called antibody and dye conjugates, which can be used for intraoperative delineation of the disease focus by selective visualization of the edge area of a disease focus. Accordingly, it is the object of this invention to prepare antibody and dye conjugates for intraoperative visualization of tumor borders. The antibodies of the antibody and dye conjugates according to the invention are directed against structures that are specific for the process of angiogenesis. The antibody and dye conjugates according to the invention comprise dyes that allow optical visualization by their concentration. Since angiogenesis becomes stronger in the area of the edge of the focus of the disease, it results there in the maximum optical signal. The antibody and dye conjugates according to the invention are suitable for visualizing the boundaries of a disease focus, which is known as the edge area, for healthy tissue by intraoperative optical diagnosis. As a result it is possible to completely remove the focus of the disease leaving the healthy tissue largely unaffected. Antibodies directed against molecules that are strongly expressed in angiogenically active tissue and that are expressed only at a very low level in the adjacent tissue are known (WO 96/01653). Antibodies directed against receptors for vascular growth factors, receptors on endothelial cells to which inflammation mediators bind, receptors on endothelial cells to which matrix molecules and matrix proteins bind are of special interest in antibody and dye conjugates. which are specifically expressed in the formation of new vessels (Brekken et al., Cancer Res. (1998) 58: 1952-9 and Schold, SC Jr. et al., Invest. Radiol. (1993) 28: 488-96) . Antibodies or antibody fragments that are directed against the fibronectin EDB matrix protein are preferred. EDB fibronectin (EDBFN) also known as oncofetal fibronectin is a fibronectin-cutting variant that specifically forms vessels of recent formation in the process of angiogenesis. The special advantage of the antibodies against fibronectin EDB is that it does not result in a new formation of fibronectin EDB in healthy tissue due to intraoperative injury during the removal of the focus of the disease. In connection with this, specificity is preserved during the surgical procedure. However, antibodies to growth factor receptors or inflammation mediators in endothelial cells that are also specifically expressed in the area of the edge of the tumor can be formed again during surgery even in healthy tissue near the focus of the disease . Especially preferred in the antibody-dye conjugate according to the invention are antibodies L19 and E8 against fibronectin EDB (Viti, F., et al., Cancer Res. (1999) 59: 347-352). These antibody and dye conjugates are also the object of this invention. The known antibodies are conjugated with dyes whose concentration in the tissue can be detected optically and allows intraoperative delineation of the edge area of a disease focus. The advantage of the antibody and dye conjugates according to the invention now consists in the bed that the latter can be used for selective fluorescent staining of tissues in a neo-angiogenic stage. Fluorescent staining is tumor specific and produces a fluorescence signal that can be detected with a high signal-to-background ratio (noise). The antibody and dye conjugates to provide fluorescence imaging are also known for the purposes of visualization of non-invasive percutaneous tumors (Neri)., D. et al., Nature Biotechnology (1997) 15: 1271-1275). However, antibody and dye conjugates that preferentially accumulate in the border area of a disease focus are not known. Protein and dye conjugates are also known for the intraoperative visualization of tumors. The disadvantage with respect to these conjugates is that it is especially the hypoxic and metabolically malnourished tumor cells that absorb the conjugates. However, as the tissue in the edge area of the tumors is well vascularized, and in this context the cells are adequately supplied with oxygen and nutrients, it is for this reason that adequate accumulation of protein and dye conjugates is not possible. known. However, the antibody and dye conjugates according to the invention are largely independent of the metabolic state of the disease focus. Although the optical detection of the boundaries of a disease focus can be carried out in different ways, it is generally preferred to detect dye-specific fluorescent radiation induced by a corresponding light stimulus. Depending on the emission wavelength in this case, the fluorescence can be detected visually in a macroscopic or microscopic direct manner, and optionally be simultaneously digitally recorded by means of image detection systems and displayed on a screen. The fluorescence radiation in the spectrum range of 400 to 650 nm is visually detectable. A wavelength of 450 to 600 nm is particularly preferred. The special advantage of using the visible light range is the fact that it allows the detection of fluorescence by a small technical expense. The stimulating light produced by laser or suitable laser diodes is coupled to a fiber optic light guide and is introduced by the latter to the area to be diagnosed. The implementation of the intraoperative detection of the edge of the tumor is carried out by means of large-scale radiation of the area. The reflected stimulating light is blocked by a filter (for example, a pair of filtering lenses worn by the person doing the study) and only the specific fluorescence of the dye is observed (macroscopic observation). As an alternative the detection of fluorescence can be carried out by means of an operating microscope (microscopic observation). Due to the small penetration depth of the VIS light in the tissue (a few millimeters), new vascular formations located on the surface can be detected in this way. Another advantage of the spectrum range of visible light exists because of the little depth of tissue penetration and tissue emission. Consequently, the detectable signal is not distorted by signals from deeper portions of the tissue and can be specifically assigned to the tissue structures that are visible on the surface. Accordingly, also subject matter of this invention are antibody and dye conjugates whose dyes induce an optical signal in the range of the visible light spectrum. The use of antibody and dye conjugates with dyes that absorb in the range of the near infrared light spectrum (NIR for its acronym in English; 600-900 nm) however allows the detection of new vascular formation in deeper layers of the tissue (up to 1 cm), since the light in the near infrared range is absorbed more weakly by the tissue and therefore has a greater depth of penetration to the tissue. The observation of fluorescence is visually impossible and can be done by CCD cameras (charge-coupled device as it is known in English) that are placed on the tissue area of interest. Both macroscopic and microscopic detection are possible. The advantage of using dyes that absorb and emit fluorescence in the range of the near infrared spectrum in the antibody and dye conjugates then becomes effective if an evaluation of masked areas (for example, by blood) is required. From the photophysiological point of view, dyes that have a maximum absorption within the range of the spectrum of 400 to 800 nm and a minimum of a maximum fluorescence within 500 to 900 nm are suitable for the antibody and dye conjugates. Objects of this invention are also antibody and dye conjugates which are characterized in that the dye induces a fluorescence signal only with the use of a defined range of wavelength of visible or near infrared light. Antibody and dye conjugates comprising visually detectable fluorescence dyes are, for example, those of the following classes: fluorescein, fluororescein-isothiocyanate, carboxyfluorescein or calcein, tetrabromofluoresceins or eosins, tetraiodofluoresceins or erythrosines, difluorofluorescein, such as, for example, Oregon Green® 488, Oregon Green® 500 or Oregon Green® 514, carboxyrodolite dyes (Oregon Green®) (US 5,227,487; US 5,442,045), carboxy amine dyes (for example, Rhodamine Green® colorants) (US 5,366,860), 4, 4-difluoro-4-bora-3a, 4a-diaza-indacene, such as, for example, Bodipy FL, Bodipy 530/550 493/503 or Bodipy and derivatives thereof (US 4,774,339, US 5,187,288, US 5,248,782, US 5,433,896 and US 5,451,663), cyanine dyes, including carbocyanines and merocyanines, coumarin dyes, such as, for example, 7-amino-4-methylcoumarin, metal complexes of DTPA or tetraatzamacrocicleno (cyclen, picleno) with dyes terbium or europium or tetrapyrrolo, in particular forfriñas. The antibody-dye conjugates comprising dyes are near infrared, for example, those of the following classes: polymethine dyes such as dyes dicarbocianina, tricarbocyanine, merocyanine oxonium (WO 96/17628), rhodamine dyes dyes fenoxacin or phenothiazine, tetrapyrrolo dyes, especially benzoporphyrins, corines and phthalocyanines. Near-infrared dyes which are preferred in the antibody-dye conjugates are cyanine dyes with absorption maxima of between 700 and 800 nm, in particular indodi and indotricarbocyanines. In the antibody and dye conjugates, dyes of the aforementioned classes having one or more carboxyl groups which are coupled to amino groups of antibodies or antibody fragments after chemical activation are generally preferred. Also preferred are those derivatives containing maleimido or bromoalkyl radicals, so that a covalent coupling to the sulfhydryl group of the amino acid cysteine is carried out. Additionally, dyes having isothiocyanate groups that also react with amino groups are preferred. In addition, the dyes in the antibody and dye conjugates should have a high photostability and not fade exposed to light irradiation (photobleaching as known in English) to ensure a constant signal within the study period. Accordingly, the subject of this invention are antibody and dye conjugates which preferably accumulate in the area of the edge of the cell tissue of a disease focus and thus render the edge area of the disease focus optically detectable. Particularly objects of this invention are the antibody and dye conjugates of general formula I B- (F) n (I) wherein B represents an antibody or an antibody fragment with high binding to ED-BFN, F represents a dye class of coumarins, fluoresceins, carboxyfluoresceins, difluorofluoresceinas, tetrabromofluoresceinas, tetayodofluoresceinas, rhodamines, carboxirodaminas, carboxirodolitas, 4, 4-difluoro-4-bora-3a, a-diaza- indacenes, polymethine dyes or dyes or complex tetrapirrolos of terbium or europium with DTPA or cyclen and its derivatives, and n represents 1 to 5. Particularly preferred and therefore are also objects of this invention the antibody conjugates and dye which dye is a cyanine dye, merocyanine dye, a dye oxonium a Styryl dye or a escuiaril dye. Especially preferred and therefore also objects of this invention are antibody and dye conjugates in which the dye portion is a cyanine dye, in particular carbocianin, dicarbocyanin or tricarbocyanin. Accordingly, the invention relates in particular to those antibody and dye conjugates in which the dye - (F) n of general formula I is a cyanine dye of general formula II where D represents a radical III or IV being that the position that is marked with a star means the point of interface with the radical B and can represent the group V, VI, VII, VIII or IX wherein R1 and R2 are sulfoalkyl-C! -C4, a saturated or unsaturated, branched or linear Cx-Cso alkyl chain, which optionally may be substituted with up to 15 oxygen atoms and / or with up to 3 carbonyl groups and / or with up to 5 hydroxy groups, R3 represents the group -C00E1, -CONE ^ 2, -NHCOE1, -NHCONHE1, - NE ^ 2, -OE1, -OSOsE1, -S03Ex, -SO? NHE1 O -E1, that E1 and E2 independent of one another represent a hydrogen atom, sulfoalkyl-C? -C, Ci-Cso saturated or unsaturated, branched or linear alkyl which optionally can be interrupted with up to 15 oxygen atoms and / or up to 3 groups carbonyl and / or can be substituted with up to 5 hydroxy groups, R4 represents a hydrogen atom or a fluorine, chlorine, bromine or iodine atom, b represents 2 or 3, X and Y represents oxygen, sulfur or the group = C ( CH3) 2 or - (CH = CH) - and L represents a direct bond or a linking agent that is a linear or branched carbon chain with up to 20 carbon atoms not that it can be substituted with one or several groups -OH, -COOH, S03 and / or optionally it can be interrupted in one or several sites by a group -O-, -S-, -CO-, -CS-, -CONH -, -NHCO-, -NHCSNH-, -S02-, -P04- or -NH or by an aryl ring. The antibody-dye conjugates according to the invention can be used either alone or in formulation as pharmaceutical agents. To use the antibody-dye conjugates as pharmaceutical agents the latter are brought into the form of a pharmaceutical preparation which in addition to the antibody and dye conjugate contains inert organic or inorganic pharmaceutical media which are suitable for enteral or parenteral administration, such as , for example, water, gelatin, gum arabic, lactose, starch, magnesium stearate, talcum, oils of vegetable origin, polyalkylene glycols, etc. The pharmaceutical preparations can be present in solid form, for example as tablets, coated tablets, suppositories, capsules or in liquid form, for example as solutions, suspensions or emulsions. Optionally they also contain adjuvants such as preservatives, stabilizers, surfactants or emulsifying agents, salts to alter the osmotic pressure or regulators. Injectable solutions or suspensions, in particular the aqueous solution of antibody and dye conjugates, are suitable for parenteral use. As vehicle systems, surfactant auxiliaries such as bile acid salts or phospholipids of animal or vegetable origin can be used, but also mixtures thereof as well as liposomes or their components. For oral use, in particular tablets, coated tablets or capsules with talc vehicles and / or hydrocarbons or binders, such as, for example, lactose, corn starch or potato are suitable. The application can also be carried out in a liquid form, such as, for example, juice to which optionally a sweetener is added. The dosage of the antibody and dye conjugates may vary depending on the method of administration, age and weight of the patient, type and severity of the disease to be treated and similar factors. The applicable dose of antibody and dye conjugates to detect limit areas is 0.5-1000 mg, preferably 500-200 mg, where the dose can be given as a single dose to be administered once or divided into two or more daily doses . The formulations and forms of administration described above are also objects of this invention. Accordingly, the invention also relates to pharmaceutical agents comprising one or more antibody and dye conjugates for intraoperative visualization of the edge areas of a disease focus, wherein the pharmaceutical agents are used either alone or in a mixture with solvents, regulators and / or suitable vehicles. The antibody and dye conjugates according to the invention are used in the surgical treatment of diseases dependent on angiogenesis, such as malignant tumors or metastases thereof, benign tumors, precancerous tissue changes, endometriosis, hemangiomas and ectopic pregnancies. The subject of this invention is also the use of antibody and dye conjugates and agents for the intraoperative visualization of disease foci, especially for microscopic and macroscopic intraoperative visualization of border areas of a disease focus, as well as the use of the antibody and dye conjugates for the production of an agent for surgical treatment of diseases dependent on 1 angiogenesis, such as malignant tumors or metastases thereof, benign tumors, precancerous tissue changes, endometriosis, hemangiomas and ectopic pregnancies. Production of the dyes The production of the dyes is carried out according to methods that are known in the literature. The dyes suitable for the production of the antibody and dye conjugates are dyes of the carboxyl groups or isothiocyanate groups for the covalent coupling to amino groups of the antibody. In this case, cyanine dyes are particularly preferred (Mujumdar, S. R. et al. (1996) 7: 356-362; Flanagan, J. H. et al. (1997) 8: 751-756 and Licha, K. et al (1996) Proc SPIE Vol. 2927, 192-198). Dyes with dyes with carboxyl groups are first activated by conversion to a reactive ester (e.g., N-hydroxysuccinimide ester) according to methods known in the art. The dyes with isothiocyanate groups can be used directly. The reactive derivatives are then reacted in a buffer solution or mixtures of organic solvent (for example, dimethylformamide (DMF) or dimethylsulfoxide (DMSO)) and buffer with the antibody. In this case, a molar excess of the dye is used from 3 to 100 times. The unreacted portion is separated by ultrafiltration and / or chromatography after the reaction has ended. The following dye is also produced by a similar method: Production example 1 N-hydroxysuccinimide ester of bis-l, l '- (4-sulfobutyl) indocarbocyanine-5-carboxylic acid The production of this acid bis-l, l' - (4-sulfobutyl) indocarbocyanine-5-carboxylic acid is carried out from 1- (4-sulfobutyl) -2,3,3-trimethyl-3H-indolenine and 1- (4-sulfobutyl) -2, 3, 3-trimethyl-5-carboxy-3H-indolenine (Cytometry 10, 11-19, 1989, Talanta 39, 505-510, 1992) in a manner similar to the methods that are known in the literature. For conversion to the N-hydroxysuccinimide ester, 0.1 mmol of the dye (67 mg in 10 ml of DMF) is mixed in each case with 0.5 mmol of N-hydroxysuccinimide and dicyclohexylcarbodiimide (DCC) and stirred for 24 hours at room temperature. After 50 ml of ether are added, the precipitated solid is separated by filtration, dissolved again twice in a little DDF each time and precipitated with ether and finally dried under vacuum (89% yield). Production of antibody-dye conjugate Production of a bis-1, 1 '- (4-sulfobutyl) -indocarbocyanine conjugate with an L19 antibody The L19 antibody (1 mg in 1 ml of sodium acetate buffer (50 mmol, pH 8.2)) is mixed with the N-hydroxysuccinimide ester (75 μmol of a 4 mg / ml solution in DMSO) and stir for 2 hours at room temperature. The purification is carried out using gel filtration in PD10 cartridges (Pharmacia) and the concentration is carried out using centricon-10 tubes (Amicon), whereby a solution of approximately 1 mg / ml of antibody is obtained. Maximum absorption: 555 nm Maximum fluorescence: 582 nm. The following example explains the biological utility of the antibody and dye conjugates according to the invention without limiting them to this exemplary application. Exemplary application 1 In vivo fluorescent imaging of nude tumor-bearing mice and ex vivo microscopic examination of tumor tissue The imaging properties of the compounds according to the invention were examined in vivo after injecting them into nude mice carrying the tumor. tumors For this purpose 0.1 μmol / kg was administered intravenously at 2 μmol / kg of the substance and the concentration in the tumor region was observed for a period of 0 to 48 hours. The fluorescence of the substances is induced by irradiation of the animals with light of a corresponding wavelength, which is produced monochromatically with a laser (laser diode, solid state laser) or filtered through a filter from the polychromatic emission of an Hg or Xe lamp. In the case of the compound described in production example 1, the light of a Nd: YAG laser of 540 nm wavelength is used to excite the stimulation of the test animal, and the fluorescence radiation to a length of wave of > 580 nm is detected by an intensified CCD camera while full-body fluorescent images are obtained. In parallel, the fluorescence is detected visually and photographically. Sections of the tumor material are prepared and studied by microscope (Zeiss Axiovert microscope with Cy3 filter set).

After the injection of 1 μmol / kg of the antibody and dye conjugate into nude mice bearing the F9 teratocarcinoma mentioned in the production example it was possible to detect after four hours an increase of the fluorescent signal compared to normal tissue based on fluorescent images of the whole body. After the preparation of the skin and the upper tissue layers of the tumor, the fluorescence can be associated with the areas of the edge of the tumor. The microscopic evaluation of the tumor sections produces a high fluorescence that correlates with blood vessels in the area of the edge of the tumor.

Claims (13)

1. CLAIMS 1. Antibody and dye conjugates that are preferably concentrated in the edge area of the cell tissue of a disease focus and thereby allow to visualize the edge area of the disease focus, characterized in that the dye is a compound of general formula I B- (F) n (I) in which B represents an antibody or an antibody fragment with high binding to fibronectin EDB, F represents a dye of the class of coumarins, fluoresceins, carboxyfluoresceins, difluorofluoresceins, tetrabromofluoresceins, tetayodofluoresceins , rhodamines, carboxyamines, carboxyrodolites, 4,4-difluoro-4-bora-3a, 4a-diaza-indacenes, polymethine dyes or tetrapyrrolo dyes or the terbium or europium complexes with DTPA or cyclin and its derivatives, and n represents 1 to 5. Antibody and dye conjugates according to claim 1, characterized in that the dye is a cyanine dye, a merocene dye, a dye of oxonium, a styryl dye or a escuarylium dye. 3. Antibody and dye conjugates according to claim 1, characterized in that the dye is a cyanine dye such as carbocyanine, dicarbocyanine or tricarbocyanine. Antibody and dye conjugates according to claims 1 to 3, characterized in that the dye - (F) n of general formula I is a cyanine dye of general formula II where D represents a radical III or IV being that the position that is marked with a star means the point of interface with the radical B and can represent the group V, VI, VII, VIII or IX wherein R x and R 2 are sulfoalkyl-C? -C 4, a saturated or unsaturated, branched or linear C 1 -C 6 alkyl chain, which optionally may be substituted with up to 15 oxygen atoms and / or with up to 3 carbonyl groups and / or with up to 5 hydroxy groups, R3 represents the group -COOE1, -CONExE2, -NHCOE1, -NHCONHE1, - NEXE2, -OE1, -OSOTÍE1, -SOSE1, -SO ^ HE1 O -E1, where E1 and E2 independent of one another represents a hydrogen atom, sulfoalkyl-C? -C, C -C50 saturated or unsaturated, branched or linear C-C50 alkyl which optionally may be interrupted with up to 15 oxygen atoms and / or up to 3 carbonyl groups and / or it can be substituted with up to 5 hydroxy groups, R4 represents a hydrogen atom or a fluorine, chlorine, bromine or iodine atom, b represents 2 or 3, X and Y represents oxygen, sulfur or the group = C (CH3) 2 or - (CH = CH) - and L represents a direct bond or a linking agent that is a linear or branched carbon chain with up to 20 carbon atoms rbonone which can be substituted with one or several groups -OH, -COOH, S03 and / or optionally can be interrupted at one or several sites by a group -O-, -S-, -CO-, -CS-, -CONH -, -NHCO-, -NHCSNH-, -S02-, -P04- or -NH or by an aryl ring. 5. Antibody and dye conjugates according to claims 1 to 4, characterized in that antibodies L19 and E8 are used as antibodies. 6. Antibody and dye conjugates according to claims 1 to 5, characterized in that the dye induces an optical signal in the range of the visible spectrum of the light. 7. Antibody and dye conjugates according to claims 1 to 5, characterized in that the dye induces a fluorescent signal only by using a certain wavelength range of visible or near infrared light. 8. Pharmaceutical agent comprising one or more antibody and dye conjugates according to claims 1 to 7 for intraoperative visualization of the edge area of a disease focus. 9. Pharmaceutical agent according to claim 8, in admixture with suitable solvents, regulators and / or vehicles. 10. Use of the antibody and dye conjugates and agents according to claims 1 to 9 for the intraoperative visualization of disease foci. 11. Use of the antibody and dye conjugates according to claims 1 to 9 for the intraoperative visualization of the border areas of a disease focus. 12. Use of the antibody and dye conjugates according to claims 1 to 9 for intraoperative micro and macroscopic visualization of the border areas of a disease focus. 13. Use of the antibody and dye conjugates according to claims 1 to 7 for the preparation of an agent for the surgical treatment of diseases dependent on angiogenesis, such as malignant tumors, their metastases, benign tumors, precancerous changes in tissues, endometriosis, hemangiomas and extrauterine pregnancies.